Method for making a biochip and biochip

ABSTRACT

The present invention relates to a method to produce a biochip and to a biochip, said biochip being composed particularly of biological probes grafted onto a conductive polymer. 
     The method according to the invention comprises the following steps: 
     a) structuring of a substrate so as to obtain on said substrate microtroughs comprising in their base a layer of a material capable of initiating and promoting the adhesion onto said layer of a film of a pyrrole and functionalised pyrrole copolymer by electropolymerisation, 
     b) collective electropolymerisation, so as to form an electropolymerised film of a pyrrole and functionalised pyrrole copolymer on the base of said microtroughs, 
     c) direct or indirect fixation of functionalised oligonucleotides by microdeposition or a liquid jet printing technique.

FIELD OF THE INVENTION

The present invention relates to a method to produce a biochip and to abiochip, said biochip being composed particularly of biological probesgrafted onto a conductive polymer.

Biological analysis devices, for example DNA chips, representhigh-performance tools for the parallel analysis of a large number ofgenes or DNA or RNA sequences. Their operating principle is based on thehybridisation or pairing property of two strands of complementarysequences in order to reconstitute the DNA double helix. To do this,oligonucleotide probes of a known sequence, immobilised on a supportsubstrate, are placed in the presence of targets extracted from abiological specimen under analysis, and labelled using fluorescentmarkers.

The hybridisation is then identified and the sequence detected byanalysing the surface of the chip with a suitable marker for example todetect the sequence by fluorescence.

Very different technologies have been used to produce these probematrices. Various immobilisation or grafting techniques of probes ontodifferent substrates have been the subject of significant studies andindustrial developments.

1. State of the Related Art

There are essentially three chemical probe addressing methods whichrepresent different approaches to the production and use of probes fordifferent fields of application. They consist of photochemicaladdressing, mechanical addressing, for example by micropipetting using adispersion robot, and electrochemical addressing.

For example, electrochemical addressing may be used for oligonucleotideprobes. To do this, individually addressed electrode matrices areproduced on a glass substrate.

The biological probe immobilisation principle is based on theelectropolymerisation deposition of a copolymer of pyrrole and pyrrolesubstituted by an oligonucleotide (Py-ODN), comprising anoligonucleotide grafted onto a pyrrole nucleus either directly, orindirectly by means of a spacer.

In order to develop massively parallel biological analysis systems, witha high capacity or active site density, it is necessary to be able toaddress a large number of probes.

Methods using electrochemical addressing require both a large electrodeand connection matrix and a multiplexer to index each of the chip'ssites electrically. In addition, in these methods, it is necessary tocarry out electropolymerisation by immersing the entire chipsuccessively in solutions of each of the Py-ODNs contained in the cell.Therefore, these methods are limited to low-density chips, i.e.comprising approximately one hundred probes, for limited and specificapplications.

Other methods have been described in the prior art, advantageouslyreplacing individual electrical addressing by mechanical addressing.However, a disadvantage remains, that of carrying outelectropolymerisations in microtroughs, with a solution volume of theorder of one nanolitre, for which it is necessary to delay evaporationafter micropipetting of all the probes on the insert so thatelectropolymerisation may take place.

2. Description of the Invention

The aim of the present invention is specifically to solve theabove-mentioned problems by providing a method to produce a biochipcomposed particularly of biological probes grafted onto a conductivepolymer, said method particularly offering the advantage of onlyrequiring the use of a single solution of a mixture of suitableproportions of pyrrole and substituted pyrrole (Py and Py-R-F or F and areactive chemical function and R is an aliphatic or aromatic spacergroup) for a single collective electrodeposition on all themicrotroughs.

The method according to the invention is characterised in that itcomprises the following steps:

a) structuring of a substrate so as to obtain on said substratemicrotroughs comprising in their base a layer of a material capable ofinitiating and promoting the adhesion onto said layer of a film of apyrrole and functionalised pyrrole copolymer by electropolymerisation,

b) collective electropolymerisation, so as to form an electropolymerisedfilm of a pyrrole and functionalised pyrrole copolymer on the base ofsaid microtroughs, on the layer of said material, using a pyrrole andfunctionalised pyrrole solution, in the presence of suitable chemicalreagents for said electropolymerisation,

c) direct or indirect fixation of a biological probe onto thefunctionalised pyrrole, by injecting a biological probe solution, eitherin one or more microtroughs in the presence of chemical reagentsrequired for the direct or indirect fixation of this biological probeonto the functionalised pyrrole.

According to the invention, the layer of material capable of initiatingand promoting the adhesion of a film of a pyrrole and functionalisedpyrrole copolymer by electropolymerisation onto said layer may be ametallic layer, step a) mentioned above possibly comprising a depositionstep of said metallic layer onto the substrate, and a deposition step ofa layer of resin or polymer onto the metallic layer and development orengraving of said layer so as to form microtroughs, wherein the base iscomposed at least partly of the metallic layer.

According to the invention, the metallic layer may be, for example, alayer of gold, a layer of copper or silver or aluminium.

According to the invention, the substrate may be for example a siliconinsert, a glass insert or a flexible plastic support if required.

According to another embodiment of the present invention, the step a)may also comprise a treatment step of the gold layer at the base of themicrotroughs in the presence of a functionalised pyrrole for examplewith a thiol group so as to form a monolayer of pyrrole onto saidmetallic layer, for example on said gold layer, at the base of saidmicrotroughs. This monolayer is capable of initiating and promoting theadhesion of a polypyrrole film by electropolymerisation as demonstratedby R. Simon et al., J. Am. Chem. Soc., 1982, 104, 2031). This is aself-assembled monolayer SAM of a functionalised pyrrole for itsadhesion onto the base of the microtroughs.

According to the invention, the functionalised pyrrole may be a pyrrolewhich comprises a chemical group enabling its fixation by covalentbonding with the metallic layer, and/or with the biological probe. Inthe case of its fixation to the metallic layer, for example to the goldlayer, a functionalised pyrrole with a thiol or disulphide group mayalso be used.

For example, the functionalised pyrrole with a thiol group may have thefollowing chemical formula:

wherein n may have a value ranging from 1 to 10, for example n may beequal to 6.

For a metallic aluminium probe, a functionalised pyrrole with a —COOHgroup may be chosen.

According to another embodiment of the present invention, the substratemay be a silicon insert and the layer capable of initiating andpromoting the adhesion onto said layer of a polypyrrole film byelectropolymerisation may be a layer of silane comprising an alignmentof pyrrole sites. Step a) of the method according to the presentinvention may in this case comprise a deposition step of a layer ofresin on the silicon insert, said silicon insert being coated with anSiO₂ film, and engraving of said resin layer so as to form themicrotroughs wherein the base is composed at least partly of the SiO₂film; and a microtrough treatment step by means of a functionalisedsilanisation agent with a pyrrole so as to fix, on the SiO₂ film, in thebase of the microtroughs, the silane layer comprising an alignment ofpyrrole sites.

According to the invention, the silanisation agent may be chosen in agroup comprising N-(3-(trimethoxy silyl) propyl) pyrrole, or any otherfunctionalised pyrrole with an —SiCl₃ or —Si(OMe)₃ group. The SiO₂ filmmay be a natural SiO₂ film present on silicon inserts.

According to the invention, irrespective of the embodiment, the resinmay be a photosensitive resin, wherein masking, insolation anddevelopment are used to form the microtroughs.

According to the invention, the collective electropolymerisation in stepb) of the method may be carried out for example by immersing thestructured substrate obtained in step a) mentioned above in anelectrolytic bath comprising a solution of pyrrole, functionalisedpyrrole, and suitable chemical reagents for electropolymerisation, inthe presence of a counter-electrode to the working electrode which isimmersed in the electrolytic bath and is independent of the structuredsubstrate.

According to the invention, in this step b), the functionalised pyrrolemay be a pyrrole comprising a group chosen in a set comprising an NH₂group, a thiol group a succinimide ester group, a trimethoxy silylgroup, a carboxyl, aldehyde and isothiocyanate group.

According to the invention, the functionalised pyrrole byelectropolymerisation may for example be chosen from one of thefollowing compounds:

PYRROLE

N-ETHYLAMINE PYRROLE

N(3-(TRIMETHOXY SILYL) PROPYL) PYRROLE

Functionalised PYRROLE with a thiol

Functionalised PYRROLE in 3′ by a succinimydyl ester.

According to the invention, the electrolytic bath may be a mixture ofpyrrole and functionalised pyrrole in suitable proportions to form afilm comprising a required number of units of functionalised pyrrole. Inthis way, the method according to the invention makes it possible tochoose the number of biological probes per microtrough, since accordingto this method, the biological probes are fixed, either directly orindirectly on said functionalised pyrroles.

The next step c) of the method according to the invention consists of adirect or indirect fixation of a biological probe onto thefunctionalised pyrrole.

According to the invention, when the fixation of the biological probe isindirect, the step c) of the method according to the invention may alsocomprise, before the fixation of the biological probe, a collectivefixation of a cross-linking agent on the functionalised pyrrole, in thepresence of suitable chemical reagents, said cross-linking agentcomprising a first function enabling its fixation onto thefunctionalised pyrrole, and a second function enabling the fixation ofthe biological probe on said cross-linking agent.

According to the invention, the cross-linking agent may for example be abi-functional cross-linking agent.

The cross-linking agent may for example comprise an N-hydroxysuccinimideester function and a maleimide function.

According to the invention, the cross-linking agent may for example bechosen from one of the following compounds;

N-hydroxysuccinimide ester maleic function

SMPB

succinimidyl 4-(p-maleimidophenyl)butyrate

GMBS

N-maleimidobutyryloxy succinimide ester,

a dialdehyde of the type

GLUTARALDEHYDE,

a diisothiocyanate of the type

1,4-PHENYLENE DIISOTHIOCYANATE,

SUCCINIC ANHYDRIDE OR SUCCINIC ACID

or a derivative of these compounds.

All the bi-functional cross-linking agents mentioned above are suitablefor functionalised polypyrroles with the —CH₂—CH₂—NH₂ group in position1 on nitrogen. However, electropolymerisation with a functionalisedpyrrole with other groups is also possible. For example, Py-CH₂—CH₂—NH₂,Py-SH, Py-succinimidyl ester (in 3), Py-hydrazine with a substitution in1 on nitrogen or in 3 on the pyrrole cycle, making it possible toimmobilise the oligonucleotides, either directly or by means of across-linking agent, for example a bi-functional agent.

The following cross-linking agents may therefore be used in the methodaccording the present invention:

a) a glutaraldehyde type dialdehyde, which may react on the NH₂ functionof the polypyrrole film (collective step) and then on the NH₂ functionof an oligonucleotide terminated for example by a phosphate comprisingan amino group, by an individual step in the microtroughs;

b) a diisothiocyanate which may also react on the amine function of thefunctionalised polypyrrole at one end (collective step) and then on anamine function of an oligonucleotide terminated by a phosphate with afunctionalised spacer group with NH₂;

c) a succinic anhydride, which for each opening, comprises two acidfunctions capable of reacting on the NH₂ groups of the polypyrrole andon the NH₂ groups of a functionalised oligonucleotide with NH₂.

According to the invention, the biological probe which will be thesource of the specificity of the manufactured biochip, may be chosen forexample from an oligonucleotide, a DNA, an RNA, a peptide, a glucide, alipid, a protein, an antibody, an antigen.

According to the invention, the biological probe is preferentiallyfunctionalised to be able to be fixed either directly or indirectly onthe functionalised pyrrole. The purpose of this functionalisation is tofix on the biological probe a chemical group capable of forming acovalent bond between the biological probe and the functionalisedpyrrole.

It may be for example functionalised with a thiol group, with an NH₂group, aldehyde, a —COOH group or an acid phosphate group.

For example, when the biological probe is an oligonucleotide, it may befunctionalised with a thiol group SH. The functionalisedoligonucleotides with S—H may be prepared according to a knownprocedure, for example at the end of an automated oligonucleotidesynthesis.

If it is easier to use functionalised oligonucleotides with NH₂, it ispossible for example to synthesise a functionalised pyrrole with an S—Hfor copolymerisation, to use for example SMPB with its two specificfunctions and immobilise the functionalised oligonucleotides with NH₂ bycovalent bonding with the succinamide function of this cross-linkingagent.

In the case of oligonucleotides terminated in 3′ by an N-methyl uridinenucleotide, an oxidation reaction on this function makes it possible toobtain a functionalised oligonucleotide with an aldehyde function,capable of reacting directly, i.e. for example without the bi-functionalagent on the functionalised polypyrrole with NH₂.

To functionalise an oligonucleotide with an NH₂ function, one of themethods that may be used according to the method of the presentinvention may consist of coupling the oligonucleotide and commerciallyavailable N-trifluoroacetyl-6 amino hexyl-2 cyanoethyl NN′-diisopropylphosphoramidite.

In addition, a functionalised oligonucleotide with NH₂ may for examplebe converted into an oligonucleotide terminated by a thiol with areaction with dithiobis(succinimidylpropionate).

The functionalised probe oligonucleotides may for example be taken up bymicropipetting in microwells and injected into the microtroughs forexample by means of a dispensing microrobot or by jet printing. Thesedevices are well-known to those skilled in the art.

The method according to the present invention makes it possibleadvantageously to choose the number of probes per active site, i.e. permicrotrough by adjusting the proportion of functionalised pyrrole withreference to the pyrrole.

The required probe density may be monitored for example by fixingoligonucleotides labelled at the chain ends by a biotin and usingstreptavidine-Cy3 detection by a surface analysis of the chip usingconventional fluorescence detection methods.

Another advantage of the method according to the invention lies in thefact that both collective operations, electropolymerisation and fixationof the cross-linking agent if applicable, may be carried out in batcheson a large number of inserts in parallel.

The inserts having undergone step a) and b) of the method according tothe invention are also referred to as “blank biochips”. They are readyto undergo the direct or indirect fixation step of a biological probe,for example of an oligonucleotide according to the present invention.

In this way, the method according to the invention makes it possible forexample to produce an oligonucleotide chip comprising in this order:

a silicon substrate coated in silica, and a functionalised silane layerwith pyrrole, or

a gold layer or a silane layer comprising pyrrole sites, or

a gold layer with or without an electropolymerisation promotion andadherence layer (based on a functionalised pyrrole with an —SH thiol),or

an aluminium layer with a functionalised pyrrole with a —COOH,

and a resin layer wherein microtroughs have been produced such that thebase of said microtroughs is composed at least partly of the gold layeror the silane layer comprising pyrrole sites,

and a layer of pyrrole and functionalised pyrrole copolymer, fixed onthe gold layer or the silane layer comprising pyrrole sites forming thebase of said microtroughs, the functionalised pyrrole being bound to abi-functional cross-linking agent or not,

and an oligonucleotide fixed directly on the functionalised pyrrole, orindirectly on the functionalised pyrrole by means of the cross-linkingagent bound with the pyrrole.

The present invention's other advantages and characteristics will beseen more clearly upon reading the following description, which isnaturally given as an illustration and is not restrictive, withreference to the appended figures.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a diagram of a section view of a structured substrateaccording to a first embodiment of steps a) and b) of the methodaccording to the present invention.

FIG. 2 is a diagram of a section view of a structured substrateaccording to the embodiment represented in FIG. 1, and also comprising across-linking agent for indirect fixation of a biological molecule.

FIG. 3 is a diagram of a section view of a structured substraterepresented in FIG. 2, illustrating the indirect fixation of anoligonucleotide on the cross-linking agent.

FIG. 4 is a diagram of a section view of a structured substrateaccording to a second embodiment of the method according to the presentinvention.

FIG. 5 is a diagram of a section view of a structured substrateaccording to a third embodiment of steps a) and b) of the methodaccording to the present invention.

EXAMPLES Example 1

Production of a biochip particularly composed of oligonucleotidesgrafted onto a conductive polymer according a first embodiment of thepresent invention.

According to this first embodiment, particularly relating to step a) ofthe method according to the invention, a gold layer is deposited on asilicon insert so as to form a working electrode for theelectropolymerisation of a pyrrole and functionalised pyrrole copolymer.Said gold layer is deposited using a conventional vacuum evaporation orcathodic pulverisation technique. It has a thickness of approximately1000 to 5000 Å and forms the collective working electrode.

A photosensitive resin is deposited on the gold electrode and aphotolithography step makes it possible to make openings in the resin soas to form microtroughs comprising the working electrode in their base,said microtroughs may be addressed simultaneously.

The resin used is preferentially:

a) a positive type photosensitive resin (Novolaque+diezonaphthoquinonedeveloping in alkaline medium);

b) a Polyimide type negative photosensitive resin (OLIN) developing inan organic solvent;

c) or a polymer engraved by dry or wet engraving.

The microtroughs formed are 100×100×30 μm in size.

The resin is deposited on the gold electrode using a conventionalspinning centrifugation technique. A structured substrate according tostep a) of the method according to the present invention is obtained inthis way.

The collective electropolymerisation step b) is carried out using apyrrole and functionalised pyrrole solution.

In this example, the functionalised pyrrole is N-ethylaminepyrrole andthe solution used for electropolymerisation is an aqueous/ethanol oracetonitrile solution comprising 0.1 mole of pyrrole, and afunctionalised pyrrole/pyrrole molar ratio of 5% to 0.5% by weight offunctionalised pyrrole. This solution is hereafter referred to as anelectrolytic bath.

The method to obtain the functionalised pyrrole monomer with an NH₂function is easy and is described for example in I. Jurkowsky, R. Baudy,Synthesis 1981, p. 481.

The electropolymerisation is carried out by immersion in theelectrolytic bath of the structured substrate obtained above, withsuitable electrochemical reagents. These reagents are for exampleelectrolytic salts (Li⁺ClO₄ ⁻, quaternary ammonium salts, Li-toxylate,or lithium, potassium or sodium sulphonate polystyrene).

The solvents for electropolymerisation are, for example, Ca₃CN, water,ethanol and water-ethanol mixtures. The pyrrole contained in the bathshows a concentration of the order of 10⁻¹ to 10⁻³ M/l.

A platinum counter-electrode and a calomel reference electrode areimmersed in the electrolytic bath and are independent of the siliconinsert, only the working electrode is incorporated in the insertstructure.

A layer of pyrrole and functionalised pyrrole copolymer is thus formedand deposited only on the base of the microtroughs by electrodeposition.

FIG. 1 is a diagram of a section view of a substrate obtained accordingto this first embodiment of the method according to the presentinvention. In this figure, reference 1 relates to the structuredsubstrate formed in this example, composed of a silicon insert 3, a goldlayer 5 and a photosensitive resin layer 7. Reference 9 relates to theconnection of the gold layer with an electric current generator forelectropolymerisation, reference 10 to a microtrough, and references 11and 13 relates to the pyrrole (reference 11) and N-ethylamine pyrrole(reference 13) copolymer formed by electrodeposition on the gold layer 5at the base of the microtrough 10.

In this example, the biological molecule fixation step c) is an indirectfixation step. It comprises the fixation of a cross-linking agent on theNH₂ function of the N-ethylamine pyrrole electrodeposited on the base ofthe microtroughs.

The cross-linking agent used in this example is succinimidyl4-(p-maleimidophenyl) butyrate) SMPB described above.

This fixation is carried out by forming a covalent bond between the NH₂function of the functionalised pyrrole and the succinate function of theSMPB.

It is carried out by immersing the previously formed substrate in a10⁻³M SMPB solution in a solvent (dimethylformamide).

The polypyrrole formed is insoluble in this solution and in the majorityof standard solvents.

FIG. 2 is a diagram of a section view of the structured substrateobtained in this way. In this diagram, reference 1 relates to thestructured substrate represented in FIG. 1, and reference 15 relates tothe cross-linking agent SMPB. This FIG. 2 also demonstrates the reactionbetween the succinimide group of the cross-linking agent and the aminefunction of the pyrrole.

Therefore, in this example microtroughs coated with a polypyrrolecomprising a surface functionalisation, by means of SMBP, of maleimidetype reagent groups were produced.

These SMBP maleimide groups enable the fixation of the biological probeon the previously electrodeposited polypyrrole film.

The biological probe used in this example is a mixture of functionalisedoligonucleotides with an SH thiol group.

The oligonucleotides were prepared with a conventional automatedsynthesis and functionalised with a thiol group. The functionalisedoligonucleotides are taken up by micropipetting in microwells andinjected into microtroughs by means of a dispensing microrobot.

FIG. 3 is a diagram of a section view of the structured substraterepresented in FIG. 2, illustrating the fixation of the oligonucleotideon the cross-linking agent. In this figure, reference 1 relates to thestructured substrate formed in this example, references 11 and 13, as inFIGS. 1 and 2, relate to the pyrrole and N-ethylamine pyrrole copolymer,reference 15 to the cross-linking agent SMBP represented in FIG. 2 andreference 17 relates to an oligonucleotide. This FIG. 3 alsodemonstrates the reaction between the maleimide function of thecross-linking agent and the —SH oligonucleotide.

The probe density was analysed by fixation of oligonucleotides labelledwith a biotin (reference 19 in FIG. 3) and using recognition withstreptavidine Cy3 (reference 21 in FIG. 3).

The analysis was carried out using a conventional fluorescence detectionmethod, applied to the biotin/streptavidine pair.

Example 2

Production of a biochip particularly composed of oligonucleotide probesgrafted onto a conductive polymer according to a second embodiment ofthe method according to the present invention.

According to this second embodiment, particularly relating to step a) ofthe method according to the invention, a negative photosensitive resinis deposited on a silicon insert coated with a natural SiO₂ film.

As in example 1, microtroughs are then formed by photolithography suchthat the base of the microtroughs, hereafter referred to as sites, arecomposed of the silicon oxide layer.

The sites are then functionalised by silanisation: saidfunctionalisation is a collective step of the method according to theinvention, it is carried out by immersing the silicon insert comprisingpreviously formed microtroughs in a functionalised silanisation agentwith a pyrrole in a suitable solvent. The silanisation agent isN-(3-(trimethyoxysilyl)propyl) pyrrole, and the solvent is anethanol/water (95/5) mixture or toluene.

On the base of the microtroughs, or sites, a monolayer of silanecomprising a regular alignment of pyrrole sites, is obtained.

This monolayer is capable of initiating and promoting the adhesion of apolypyrrole film by electropolymerisation: it forms a working electrodefor the collective electropolymerisation of the method according to thepresent invention.

Electropolymerisation on such a monolayer is for example described inthe article by P. Simon et al., J. Am. Chem. Soc. 1982, 104, 2031.

The next step is step b) of the method according to the invention, theelectropolymerisation of a pyrrole and N-ethylamine pyrrole copolymerhereafter referred to as Py and Py-R-F, where R and F are respectively aspacer group and a reactive chemical function.

The functionalised silicon insert with silane pyrrole in fact forms theanode of an electrolytic cell. It is immersed in a suitable electrolyticbath, containing both polymers, a counter-electrode and a referenceelectrode.

The electrolytic bath also comprises Py and Py-R-F of the Li⁺electrolytic salts in a water/ethanol or acetonitrile solvent.

The counter-electrode is a platinum electrode. During theelectropolymerisation, the pyrrole and substituted pyrrole nuclei areinserted in and bound with the pyrrole units of the silane monolayer.

FIG. 4 appended illustrates the product obtained in this way and alsoshows the formation of covalent bonds between the different pyrrolecycles.

In this figure, reference 32 relates to the silicon insert, reference 34to the photosensitive resin layer, reference 35 to a microtrough,reference 36 to the silane monolayer, and reference 38 to the layer ofpyrrole and functionalised pyrrole copolymer.

The biochip is produced as in example 1:

reactions with the bi-functional cross-linking agent: collective step,

immobilisation of the functionalised oligonucleotide probes with a thiolgroup (—S—H) by mechanical addressing with a robot by GESIM type liquidjet printing (piezoelectric head) or with a BROWN type robot.

Example 3

Production of a biochip particularly composed of oligonucleotide probesgrafted onto a conductive polymer according to a third embodiment of themethod according to the present invention.

In this example of an embodiment of the method according to the presentinvention, the microtroughs were produced by photolithography of a resindeposited on a gold electrode on the surface of a silica insert as inexample 1 above.

Thiolisation of the gold layer at the base of the microtroughs was thenperformed by a functionalised pyrrole with an —SH group according to thefollowing formula:

The reaction was carried out by immersing the above-mentioned insert ina solution containing the functionalised pyrrole with a thiol in asolvent such as dimethylformamide DMH for example.

The thiol adhered on the gold at the base of the microtroughs to form apyrrole monolayer. The combined gold layer and pyrrole fixed on saidlayer form a working electrode for the collective electropolymerisationof step b) of the method according to the invention. In fact, thespecimen serves as an anode for the collective priming of theelectropolymerisation.

Steps b) and b) of the method according to the invention were thencarried out as in examples 1 and 2 above.

FIG. 5 appended is a diagram illustrating the product obtained in thisexample. It consists of a section view of a structured substrate 40comprising a silicon insert 42, a gold layer 44, a photosensitive resinlayer 46 wherein microtroughs 48 are formed, a pyrrole monolayer 50adhered onto the gold at the base of the microtroughs, and a film 53 ofpyrrole Py and

functionalised pyrrole copolymer. In this figure, the curved arrowsindicate the electrodeposition of the above-mentioned film on thefunctionalised pyrrole 50 adhered by thiol groups onto the gold at thebase of the microtroughs.

Example 4

Additional Examples

Another approach consists of using a deposition of functionalisedpolypyrrole, such as:

either an oligonucleotide immobilisation support,

or a support to start in situ oligonucleotide synthesis.

This technique makes it possible to replace advantageously asilanisation step wherein a monolayer is more difficult to produce, by apolymer film comprising a well-controlled thickness and number offunctional sites.

To do this, films of a copolymer comprising a given proportion offunctionalised pyrrole with reference to the pyrrole is produced byelectropolymerisation. These polypyrrole films, deposited on a goldelectrode, instead of silicon or glass, show a parasite fluorescence ofan intensity well below that observed with the other substrates.

The functionalisation may be carried out:

1. on the nitrogen of the pyrrole by an NH₂ or epoxy function, forexample:

These functions may serve both for the immobilisation of the probes andthe in situ synthesis;

2. in position 1, 2 or 3 of the pyrrole by an oxyamine (R—ONH₂) orcarbonyl (R, R′C=0 where preferentially R′=CH₃) function. In this case,these functions serve only for probe immobilisation. The oligonucleotidepreferentially comprises either a carbonyl function or an oxyaminefunction according to the substrate. The oxyamine-carbonyl couplingreaction offers the advantage of being very rapid and results inimmobilisation times of less than 10 minutes, compared to a few hours asin the previous case;

3. on the nitrogen of the pyrrole by a nucleotide preferentiallycomprising a T base. This functionalised serves for the immobilisationof probes comprising a psolarene group in 5′. This group reacts underthe effect of light at 365 nm to perform a cycloaddition between thedouble bond of the psolarene and the double bond 5,6 of Thymine; thereaction time is relatively short: approximately 15 min.

What is claimed is:
 1. A method of producing a blank biochip,comprising: a) providing a substrate; b) depositing a layer of materialonto a surface of said substrate; c) coating the layer of material witha resin layer; and d) producing a plurality of microwells in the resinlayer wherein the layer of material forms at least a part of the base ofthe microwells; and e) initiating and promoting on said base of themicrowells the adhesion of a copolymer film comprising pyrrole andfunctionalized pyrrole by electropolymerization after the formation ofthe microwells, wherein the copolymer film allows for the fixation of abiological probe on the base of the microwells.
 2. The method of claim1, further comprising e) directly or indirectly fixating a biologicalprobe to the functionalised pyrrole by injecting a biological probesolution, in one or more microwells in the presence of chemical reagentsrequired for the fixating.
 3. The method of claim 1, wherein the layerof material is a metallic layer and wherein b) further comprisesdepositing the metallic layer onto the substrate; depositing a layer ofresin or polymer onto the metallic layer; and engraving the resin layerto form microwells; and wherein the metallic layer forms at least a partof the base of the microwells.
 4. The method of claim 3, wherein themetallic layer is a gold layer.
 5. The method of claim 4, which furthercomprises chemically treating the gold layer at the base of themicrowells in the presence of a functionalized pyrrole to form a pyrrolemonolayer to the gold layer at the base of the microwells.
 6. The methodof claim 5, wherein the functionalized pyrrole contains a thiol group.7. The method of claim 6, wherein the functionalized pyrrole with athiol group has the following chemical formula:

wherein n is from 2 to
 10. 8. The method according to claim 1, whereinthe substrate is a silicon insert.
 9. The method of claim 1, wherein thesubstrate is a silicon insert and the layer of material is a layer ofsilane comprising an alignment of pyrrole sites; wherein the methodfurther comprises depositing a layer of resin on the silicon insert,which is coated with an SiO₂ film; and engraving the resin layer to formthe microwells, wherein the SiO₂ film forms at least a part of the baseof the microwells; and treating the microwells with a functionalizedsilanization agent and a pyrrole to fix the silane layer comprising analignment of pyrrole sites on the SiO₂ film in the base of themicrowells.
 10. The method of claim 9, wherein the silanisation agent isselected from the group consisting ofN(3-(trimethoxysilyl)propyl)pyrrole, a functionalized pyrrole with a—SiCl₃, and a functionalized pyrrole with a —Si(OMe)₃ group.
 11. Themethod of claim 1, which further comprises immersing the structuredsubstrate in an electrolytic bath comprising a solution of pyrrole,functionalised pyrrole, and suitable chemical reagents forelectropolymerisation, in the presence of a counterelectrode which isimmersed in the electrolytic bath and is independent of the structuredsubstrate, wherein the layer of material forms a working electrode. 12.The method of claim 1, wherein the functionalised pyrrole is a pyrrolewith a group selected from the group consisting of an NH₂ group, a thiolgroup, an N-hydroxysuccinimide ester group, a trimethoxy silyl group, acarboxyl group, an aldehyde group, and an isothiocyanate group.
 13. Themethod of claim 1, wherein the functionalised pyrrole is selected fromthe group consisting of:


14. The method of claim 2, wherein prior to fixating the biologicalprobe, the method further comprises collectively fixating across-linking agent on the functionalized pyrrole in the presence ofsuitable chemical reagents, wherein the crosslinking agent comprises afirst function enabling its fixation onto the functionalised pyrrole,and a second function enabling the fixation of the biological probe onthe cross-linking agent.
 15. The method of claim 14, wherein thecross-linking agent is selected from the group consisting of adialdehyde, a diisothiocyanate, a diacid, a succinic anhydride, and aderivative thereof.
 16. The method of claim 14, wherein thecross-linking agent selected from the group consisting of:


17. The method of claim 2, wherein the biological probe is selected fromthe group consisting of an oligonucleotide, DNA, RNA, a peptide, aglucide, a lipid, a protein, an antibody, and an antigen.
 18. The methodof claim 17, wherein the oligonucleotide is functionalized with a thiolgroup.
 19. The method according to claim 4, which further compriseschemically treating the gold layer at the base of the microwells in thepresence of a functionalised pyrrole to form a monolayer of pyrrole onthe gold layer at the base of the microwells.
 20. The method of claim19, wherein the pyrrole is functionalized with a thiol group.
 21. Themethod of claim 20, wherein the functionalized pyrrole with a thiolgroup has the following chemical formula:

wherein n is from 2 to
 10. 22. The method of claim 2, wherein thesubstrate is a silicon insert.
 23. The method of claim 2, which furthercomprises immersing the structured substrate in an electrolytic bathcomprising a solution of pyrrole, functionalized pyrrole, and suitablechemical reagents for electropolymerisation, in the presence of acounterelectrode which is immersed in the electrolytic bath and isindependent of the structured substrate, wherein the layer of materialforms a working electrode.
 24. The method according to claim 2, whereinthe functionalised pyrrole is a pyrrole comprising a group consisting ofan NH₂ group, a thiol group, an N-hydroxysuccinimide ester group, atrimethoxy silyl group, a carboxyl group, an aldehyde group, and aisothiocyanate group.
 25. The method according to claim 2, wherein thefunctionalised pyrrole is selected from the group consisting of:


26. A blank biochip comprising in this order: a substrate; a layer ofmaterial that can initiate and promote the adhesion of a pyrrole andfunctionalised pyrrole copolymer film on the layer of material byelectropolymerisation; a layer of resin coating the layer of material,forming microwells such that the base of the microwells is composed atleast partly of the layer of material; and a pyrrole and functionalisedpyrrole copolymer layer fixed on the base of the microwells.
 27. Abiochip comprising in this order; a silica substrate; a gold layercomprising pyrrole sites; a resin layer coating the gold layercomprising pyrrole sites forming microwells such that the base of themicrowells is composed at least partly of the gold layer comprisingpyrrole sites; a pyrrole and functionalised pyrrole copolymer layerfixed on the gold layer comprising pyrrole sites at the base of themicrowells, wherein the functionalised pyrrole is bound or not bound toa bi-functional cross-linking agent, and an oligonucleotide fixeddirectly on the functionalised pyrrole or fixed indirectly on thefunctionalised pyrrole by the cross-linking agent bound to the pyrrole.28. The method of claim 2, wherein the biological probe is afunctionalized oligonucleotide and which is fixed directly or indirectlyonto the functionalized pyrrole.
 29. A biochip comprising in this order;a silica substrate; a silane layer comprising pyrrole sites; a resinlayer coating the silane layer comprising pyrrole sites formingmicrowells such that the base of the microwells is composed at leastpartly of the silane layer comprising pyrrole sites; a pyrrole andfunctionalised pyrrole copolymer layer fixed on the silane layercomprising pyrrole sites at the base of the microwells, wherein thefunctionalised pyrrole is bound or not bound to a bi-functionalcross-linking agent, and an oligonucleotide fixed directly on thefunctionalised pyrrole or fixed indirectly on the functionalised pyrroleby the cross-linking agent bound to the pyrrole.